Oncolytic virus replicating selectively in tumor cells

ABSTRACT

By using a virus having a gene sequence comprising a telomerase promoter and an E1 gene (preferably a sequence comprising E1A gene, IRES sequence and E1B gene) or by using an anticancer agent comprising the virus, the virus replicates in cancer cells to thereby produce an efficient anticancer effect.

TECHNICAL FIELD

The present invention relates to a virus showing antitumor effect byreplicating in tumor cells; a polynucleotide contained in the virus; ananticancer agent comprising the virus; and a method of treating cancersusing the virus.

BACKGROUND ART

At present, gene therapy is performed as one method for treatingcancers. However, since a gene is introduced into diseased tissue or thelike with a non-replication competent virus vector in gene therapy, thegene can be applied to only those regions around target cells takinginto consideration the safety of the human body. Also, in the genetherapy currently practiced, satisfactory therapeutic effect cannot beachieved because of low efficiency in gene transfer.

It is known that telomerase activity is often enhanced in malignantlytransformed cells or immortalized cell strains, whereas telomeraseactivity is hardly detected in normal somatic cells excluding such asgerm line cells, blood lineage cells and epithelial stem cells.

Under circumstances, it is a major object of the present invention tolet a virus grow in tumor cells by utilizing the telomerase activatedtherein to thereby bring death to the tumor cells efficiently.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic drawing of the structure of a oncolytic virusreplicating selectively in tumor cells. A replication cassetteconsisting of hTERT promoter, E1A gene, IRES sequence and E1B gene isinserted in the E1 gene region which non-replication competent virusvectors lack.

FIG. 2 shows comparison of telomerase activities in human cancer cellsand normal cells.

FIG. 3 shows the expression of E1A and E1B mRNAs and proteins after TRADinfection in human cancer cells and normal cells.

FIG. 4 shows the intracellular replication of the virus after TRADinfection in human cancer cells and normal cells.

FIG. 5 presents photographs showing, by staining with Coomassiebrilliant blue, the cytotoxicity caused by TRAD in human cancer cellsand normal cells.

FIG. 6 presents microscopic photographs showing the cytotoxicity causedby TRAD in human cancer cells and normal cells.

FIG. 7 presents graphs showing by means of XTT assay the cytotoxicitycaused by TRAD in human cancer cells and normal cells.

FIG. 8 is a graph showing the antitumor effect produced by intratumoral,local administration of a non-replication competent, p53 gene-expressingadenovirus vector in an experiment using nude mice and human lung cancercell H358.

FIG. 9 is a graph showing the antitumor effect produced by intratumoral,local administration of TRAD in an experiment using nude mice and humanlarge bowel cancer cell SW620.

DISCLOSURE OF THE INVENTION

The present inventors have found for the first time that, by infectingcancer cells with a virus having a telomerase promoter and replicationability, it is possible to let the virus replicate in the cancer cellsand bring death to them. Thus, the present invention has been achieved.

The present invention relates to the following items 1 to 10.

1. A polynucleotide comprising a promoter from human telomerase and atleast one E1 gene.

2. The polynucleotide of item 1 above, wherein the E1 gene is anadenovirus-derived E1 gene.

3. The polynucleotide of item 1 or 2 above, wherein the promoter fromhuman telomerase is hTERT.

4. The polynucleotide of any one of items 1 to 3 above, wherein the E1gene comprises an E1A gene, an IRES sequence and an E1B gene in thisorder.

5. A virus comprising the polynucleotide of any one of items 1 to 4above.

6. The virus of item 5 above, wherein the virus is an adenovirus.

7. An anticancer agent comprising the virus of item 5 or 6 above as anactive ingredient and a pharmaceutically acceptable carrier, excipientor diluent.

8. A method of treating a cancer, comprising using the virus of item 5or 6 above or using the anticancer agent of item 7 above.

9. The method of item 8 above, wherein the cancer is at least one cancerselected from the group consisting of stomach cancer, large bowelcancer, lung cancer, liver cancer, prostate cancer, pancreas cancer,esophagus cancer, bladder cancer, gallbladder/bile duct cancer, breastcancer, uterine cancer, thyroid cancer and ovarian cancer.

10. The method of item 9 above, wherein the cancer is at least oneselected from the group consisting of osteosarcoma and brain tumor.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is characterized by bringing death to cancer cellsby infecting cancer cells with a virus having a telomerase promoter andreplication ability and letting the virus grow in the cancer cells,based on the finding that a wide variety of cancer cells have telomeraseactivity.

The virus used in the present invention is not particularly limited.From the viewpoint of safety, adenovirus is preferable. Among adenovirusspecies, type 5 adenovirus is particularly preferable from the viewpointof, for example, easiness in use.

E1 gene contained in viral polynucleotide refers to one of early genesof viruses. Viruses have early (E) genes and late (L) genes involved intheir DNA replication. E1 gene encodes a protein involved in theregulation of transcription of viral genome.

The E1 gene used in the present invention may be derived from any virus.Preferably, an adenovirus-derived E1 gene is used.

It is known that E1 gene is composed of E1A, E1B and other elements. E1Aprotein encoded by E1A gene activates the transcription of a group ofgenes (E1B, E2, E4, etc.) necessary for the production of infectiousvirus.

E1B protein encoded by E1B gene assists the accumulation of late gene (Lgene) mRNA in the cytoplasm of the infected host cell to thereby inhibitthe protein synthesis in the host cell. Thus, E1B protein promotes viralreplication. The sequences of adenovirus E1A gene and E1B gene are shownin SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

In the present invention, a known E1 gene may be used as it is.Preferably, an E1 gene having an E1A gene, an IRES sequence and an E1Bgene in this order (i.e., an E1 gene in which an IRES sequence isinserted between its E1A gene and E1B gene) is used. With the use ofsuch an E1 gene, the replication ability of the virus of the inventionwill be high when a host cell has been infected with the virus.

As long as the effect of the invention can be achieved, at least onenucleotide may be inserted into at least one site selected from thegroup consisting of (a) between IRES sequence and E1A gene, (b) betweenIRES sequence and E1B gene, (c) upstream of E1A gene, and (d) downstreamof E1B gene. As long as the effect of the invention can be achieved, atleast one, preferably several nucleotides may be substituted, deleted,inserted or added in the E1A gene, IRES sequence, E1B gene or E1 gene.

“IRES sequence” is a protein synthesis initiation signal specific topicornavirus. It is believed that this sequence serves as aribosome-binding site because it contains a complementary sequence tothe 3′ terminal sequence of 18S ribosomal RNA. It is known thatpicornavirus-derived mRNA is translated via this sequence.

Translation efficiency from IRES sequence is high. Even from the middleof mRNA, protein synthesis is performed in a cap structure non-dependentmanner. Therefore, in the virus of the present invention, both E1A geneand E1B gene located downstream of the IRES sequence are translatedindependently by a promoter from human telomerase. IRES sequence isshown in SEQ ID NO: 3.

In the present invention, it is preferable that E1 gene has a promoterfrom human telomerase upstream thereof, because such a promoter iscapable of promoting the replication of the virus of the invention incancer cells having telomerase activity. The promoter from humantelomerase is not particularly limited as long as the promoter isderived from human. Among all, hTERT is preferable.

hTERT is a gene encoding human telomerase reverse transcriptase. Anumber of transcription factor-binding sequences are confirmed in a 1.4kbp region upstream of the 5′ end of this gene. This region is believedto be hTERT promoter. In particular, a 181 bp sequence located upstreamof the translation initiation site is a core region important for theexpression of the downstream gene.

In the present invention, any sequence comprising this core region maybe used as a promoter from human telomerase. Preferably, an upstreamsequence of approximately 378 bp containing the core region completelyis used. It has been confirmed that this sequence of approximately 378bp is equivalent to the 181 bp core region alone in gene expressionefficiency. The sequence of hTERT is shown in SEQ ID NO: 4.

A gene having the telomerase promoter of the invention and the E1 geneof the invention (a gene comprising E1A gene, IRES gene and E1B gene)may be obtained by conventional genetic engineering techniques.

As the E1 gene, an E1 gene from a known virus having that gene may beused. Preferably, an E1 gene derived from adenovirus is used.

Alternatively, E1A gene and E1B gene may be amplified from E1gene-expressing cells (preferably, E1 gene-expressing 293 cells or thelike) by RT-PCR and/or DNA-PCR using primers such as E1A-S, E1A-AS,E1B-S and E1B-AS. If necessary, their sequences are confined using aconventional method such as TA cloning. Then, E1A and E1B DNA fragmentsmay be cut out using a known restriction enzyme such as EcoRI.

E1A and E1B may be inserted into a known vector such as pIRES byconventional genetic engineering techniques to thereby prepareE1A-IRES-E1B sequence within the vector. Subsequently hTERT promotersequence which was cut out with restriction enzymes such as MluI andBglII may be inserted into the XhoI site or the like located upstream ofE1A.

If necessary, cytomegalovirus (CMV) promoter contained in a known vectorsuch as pShuttle may be removed with restriction enzymes such as MfeIand NheI; then, a sequence cut out from phTERT-EIA-IRES-E1B withrestriction enzymes NheI and NotI may be inserted into the site(resultant vector is designated “pSh-hAIB”).

From the resultant pSh-hAIB, a sequence comprising necessary portions(including hTERT promoter, E1A gene, IRES sequence and E1B gene) may becut out with restriction enzymes such as 1-CeuI and P1-SceI, and theninserted into a viral DNA such as Adeno-X Viral DNA using a commercialkit such as Adeno-X Expression System (Clontech) (the resultant DNA isdesignated “AdenoX-hAIB”).

The above-described sequence comprising hTERT promoter, E1A gene, IRESsequence and E1B gene may be inserted into any site of a viral gene aslong as the effect of the present invention can be achieved. Forexample, in adenovirus for gene therapy from which E1 gene has beendeleted, the above-described sequence is preferably inserted into thedeleted site.

It is possible to linearize AdenoX-hAIB with a known restriction enzymesuch as PacI and then transfect into cultured cells such as 293 cells,to thereby prepare a infectious recombinant adenovirus (the resultantvirus is sometimes called the “virus of the present invention” or“TRAD”). The method of transfection is not particularly limited. Fromthe viewpoint of efficiency, such methods as the calcium phosphatemethod or electroporation may be preferable.

The thus obtained virus of the present invention can be replicated byconventional methods for viral replication, e.g. infecting host cellssuch as 293 cells with the virus.

The virus of the present invention may be used as an anticancer agent.This anticancer agent may be used not only for treating cancers but alsofor preventing postoperative relapse of cancers, preventing cancermetastasis and/or for prophylaxis of cancers.

The kinds of cancers to which the anticancer agent of the invention isapplied are not particularly limited. The anticancer agent is applicableto any kind of cancer. For example, the anticancer agent is effectivefor cancers in the stomach, large bowel, lung, liver, prostate,pancreas, esophagus, bladder, gallbladder/bile duct, breast, uterus,thyroid, ovary, etc. as well as brain tumor and osteosarcoma. Among all,the anticancer agent is especially effective for solid tumor.

The anticancer agent of the invention may be applied to diseased sitesas it is. Alternatively, the anticancer agent may be introduced intohumans (target cells or organs) by any known method, e.g. intravenous,intramuscular, intraperitoneal or subcutaneous injection; inhalationthrough the nasal cavity, oral cavity or lung; oral administration;administration in the form of suppository; and administration in theform of external medicine.

The virus of the invention may be treated, for example, by thelyophilization method to enable easy handling and then used alone, orprepared into pharmaceutical compositions by mixing with knownpharmaceutically acceptable carriers such as excipients, fillers,binders, lubricants; or known additives (including such as buffers,isotonic agents, chelating agents, coloring agents, preservatives,fragrances, flavoring agents, and sweetening agents).

The anticancer agent of the present invention may be administered orallyor parenterally depending on the form of the agent, e.g. oraladministration agents such as tablets, capsules, powders, granules,pills, liquids, syrups, etc. and parenteral administration agents suchas injections, external medicines, suppositories, eye drops, etc.Preferably, local injection into muscle or abdominal cavity, orintravenous injection may be enumerated.

Dose levels are selected appropriately depending on the kind of activeingredient, the administration route, the target of administration, andthe age, body weight, sex, symptoms and other conditions of the patient.Usually, dose levels may be selected so that the virus of the invention(the active ingredient) is administered at a daily dose of about10⁶-10¹¹ PFU, preferably about 10⁹-10¹¹ PFU. This amount may beadministered once a day, or may be divided into several portions andadministered at several times a day.

When the virus of the invention is administered, it is also possible touse a known immunosuppressant or the like to suppress the immunity ofthe living body to thereby make the viral infection easy.

Further, the virus of the invention may be used jointly with at leastone anticancer agent selected from the group consisting ofnon-replication competent viruses (such as virus comprising p53 gene)used in conventional gene therapy, known anticancer agents andradiation.

The virus of the invention infected to the living body (cancer cells orcancer tissues) is capable of replicating in the cancer cells andbringing death to those cells. By thus bringing death to cancer cells,the virus of the invention can treat cancers, inhibit the growth oftumor cells, and prevent metastasis of cancer cells.

It is believed that there is an extremely low possibility that theanticancer agent of the invention will produce side effects for thereasons described below. Thus, the anticancer agent of the invention canbe said a very safe preparation.

(1) There is little telomerase activity in normal somatic cells, and yetadenovirus itself is hard to be infected to suspending cells such ashematopoietic cells. Therefore, when adenovirus is used in the presentinvention, still higher selectivity for tumor kinds is obtained.

(2) Since the virus of the invention has replication ability, it ispossible to use this virus at a lower concentration than that ofconventional non-replication competent virus used in conventional genetherapy.

(3) Even when the virus of the invention has been administered inexcess, antiviral action works through ordinary immune reaction in theliving body.

EXAMPLES

Hereinbelow, examples will be provided in order to illustrate thepresent invention in more detail. Needless to say, the present inventionis not limited to these examples.

Example 1

<Preparation of TRAD>

An E1A gene of 899 bp was amplified from RNA extracted from 293 cells byRT-PCR using specific primers (E1A-S: SEQ ID NO: 5; E1A-AS: SEQ ID NO:6). An E1B gene of 1823 bp was amplified from DNA extracted from 293cells by DNA-PCR using primers (E1B-S: SEQ ID NO: 7; E1B-AS: SEQ ID NO:8).

These PCR products were subjected to TA cloning (TA Cloning Kit DualPromoter; Invitrogen) to thereby confirm their sequences. Then, DNAfragments of 899 bp (E1A) and 1823 bp (E1B) were cut out, respectively,with restriction enzyme EcoRI.

E1A and E1B were inserted into the MluI site and the SalI site of pIRESvector (Clontech), respectively, in the normal orientation(E1A-IRES-E1B).

A 455 bp hTERT promoter sequence which had been cut out with restrictionenzymes MluI and BglII was inserted into the XhoI site located upstreamof the E1A of E1A-IRES-E1B (phTERT-E1A-IRES-E1B).

The cytomegalovirus (CMV) promoter contained in pShuttle vector wasremoved by treatment with restriction enzymes MfeI and NheI. Then, a3828 bp sequence cut out from phTERT-E1A-IRES-E1B using restrictionenzymes NheI and NotI was inserted into that site (pSh-hAIB).

A 4381 bp sequence was cut out from pSh-hAIB using restriction enzymesI-CeuI and Pl-SceI, and inserted into the Adeno-X Viral DNA of Adeno-XExpression System (Clontech) (AdenoX-hAIB). This AdenoX-hAIB was treatedwith restriction enzyme PacI for linearization and then transfected into293 cells by the phosphate calcium method. Thus, a infectiousrecombinant adenovirus (TRAD) was prepared. A schematic drawing of TRADis shown in FIG. 1.

Example 2

<Comparison of Telomerase Activities in Human Cancer Cells and NormalCells>

RNA was extracted from the following 10 kinds of cells using RNAzol(Cinna/Biotecx): human lung cancer cells (A549, H226Br and H1299); humanlarge bowel cancer cells (SW620, DLD-1 and LoVo); human embryonic kidneycell 293; human umbilical vascular endothelial cell HUVEC immortalizedby the introduction of SV40 gene; and human normal fibroblast cells(W138 and NHLF). The resultant RNA was subjected to real timequantitative reverse transcription (RT)-PCR using Light Cycler DNATeloTAGGG Kit (Roche Molecular Biochemicals), followed by comparison ofexpression levels of HTERT gene in respective cells. The results areshown in FIG. 2.

When expression levels were compared taking the level in A549 cells(which showed the highest expression) as 1.0, hTERT gene expression from0.18 to 1.00 was observed in cancer cells (such as A549, H226Br, H1299,SW620, DLD-1, Lovo) and 293 cells, whereas no expression was detected inimmortalized cell HuVEC and normal cells (such as W138, NHLF).

Example 3

<Expression of E1A and E1B mRNAs and Proteins after TRAD Infection inHuman Cancer Cells and Normal Cells>

Human large bowel cancer cell SW620 and human normal fibroblast cellW138 were cultured in vitro. Then, each cell was infected with TRAD atconcentrations of MOI (multiplicity of infection) 0.1 and 1, followed byrecovery of RNA after 36 hours. As a positive control, 293 cells wereused.

The recovered RNA was reverse-transcribed using GeneAmp RNA PCR CoreKit. The resultant DNA was amplified 30 cycles in GeneAmp PCR System9700 Thermal Cycler (PE Applied Biosystems) using primers for E1A geneand E1B gene. The PCR products were electrophoresed on 1.2% agarose geland stained with ethidium bromide to thereby visualize bands (upper twopanels in FIG. 3A). The intensities of the bands were measured with animage analyzer, quantitatively determined using GAPDH as an internalcontrol and then shown in graphs (the bottom panel in FIG. 3A).

Human large bowel cancer cell SW620 and human normal fibroblast cellWI38 were cultured in vitro. Then, each cell was infected with TRAD atconcentrations of MOI 0.1 and 1. After 48 hours, adherent cells wererecovered and reacted in a lysis solution for 30 minutes, followed bycentrifugation. The protein concentration in the resultant supernatantwas measured. Briefly, the supernatant was electrophoresed on 12%polyacrylamide gel and transferred onto a membrane. Then, Western blotanalysis was performed with anti-adenovirus 5 E1A antibody (PharMingenInternational). The results are shown in FIG. 3B.

While strong expression of E1A gene (502 bp) and E1B gene (543 bp) wasclearly observed as a result of TRAD infection in cancer cell SW620,only weak expression of these genes was observed in normal cell W138(FIG. 3A). In the positive control 293 cells, medium expression of thesegenes was observed.

The results of Western blot analysis revealed that expression of E1Aprotein increased in SW620 as the concentration of TRAD increased fromMOI 0.1 to 1 (FIG. 3B). On the other hand, expression of E1A protein wasdetected little in W138 even when TRAD was used at MOI 1.

Example 4

<Examination of Intracellular Viral Replication after TRAD Infection inHuman Cancer Cells and Normal Cells>

Human cancer cells (SW620 and H1299) and human normal cells (W138 andNHLF) were infected with TRAD at MOI for 2 hours at 37° C. Then, theTRAD-containing culture broth was discarded. After cells were washedwith a fresh culture broth once, a fresh culture broth was addedfurther. Immediately thereafter (i.e., on day 0), cells were recoveredwith a scraper and subjected to repetition of freezing and thawing.Then, they were suspended in 1 ml of a culture broth. Further, virus wasrecovered on day 1, 2, 3, 5 and 7 in the same manner, followed bymeasurement of viral titer. The results are shown in FIG. 4.

In normal cells W138 and NHLF, TRAD increased from 10² PFU on day 1 toabout 10⁵ PFU on day 3 showing 100- to 1000-fold growth. On the otherhand, in cancer cells SW620 and H1299, TRAD increased to 10⁷-10⁸ PFUshowing 10⁵- to 10⁶-fold growth. Thus, viral growth specific to cancercells was confirmed.

Example 5

<Cytotoxic Activity of TRAD in Human Cancer Cells and Normal Cells>

Five kinds of human cancer cells (SW7620, H1299, A549, DLD-1 and H226Br)were plated on 24-well plates at 6-8×10⁴ cells/well, and two kinds ofhuman normal cells (W138 and NHLF) were plated on 24-well plates at2-4×10⁴ cells/well. After 24 hours, they were infected with TRAD at MOI0.01, 0.1, 1, 2 and 5. Ninety-six hours after the infection,morphological changes in SW620, DLD-1 and NHLF cells were observed undermicroscopy. Further, culture broth was discarded from all of the cells.Then, viable cells were stained with Coomassie brilliant blue, andmacroscopic images were taken into with a scanner.

SW620 and H1299 were plated at 10⁴ cells/well and NHLF was plated at5×10³ cells/well, respectively, on 96-well plates. Cells were infectedwith TRAD at MOI 0 (non-infected cells), 0.01, 0.1 and 1. Then, thenumbers of viable cells were measured by XTT assay on day 1, 2, 3, 5 and7. The viable cell count was determined for each four wells. Taking thecount in the non-infected cells as 1.0, counts in other cells wererepresented in graphs in means +/−SDs. Respective results are shown inFIGS. 5, 6 and 7.

In cancer cells SW620, H1299, A549, DLD-1 and H226Br, cell countsdecrease and areas stained with blue reduce in a TRADconcentration-dependant manner. On the other hand, in normal cells W138and NHLF, no remarkable decrease in the number of viable cells stainedwith blue was recognized (FIG. 5).

In the microscopic observation, SW620 and DLD-1 cells were peeled offfrom the plate bottom, became round-shaped and showed decrease in celldensity; on the other hand, NHLF cells showed little morphologicalchange and no decrease in cell count (FIG. 6).

In SW620 and H1299 cells, almost 100% cell death was observed by day 3as a result of TRAD infection at MOI 1. More than 80% decrease in cellcount was recognized even at MOI 0.1. On the other hand, NHLF showedalmost no decrease in cell count even on day 3. Although NHLF showedabout 60% decrease in cell count on day 7 when TRAD was used at MOI 1,it indicated no viral influence at MOI 0.01 (FIG. 7).

Example 6

<Examination of the Antitumor Activity of TRAD in Animal Models>

Human lung cancer cell H1358 was transplanted subcutaneously into theback of 5-6 week-old nude mice at 5×10⁶ cells/mouse. When the tumorbecame approximately 5-6 mm in diameter, a non-replication competentadenovirus vector (Ad-p53) was injected intratumorally and locally forconsecutive two days at 1×10⁸ PFU, 3×10⁸ PFU and 1×10⁹ PFU per day.Then, two axes of each tumor crossing at right angles were measured atregular intervals. The estimated tumor weight was calculated by thefollowing formula: (major axis)×(minor axis)²/2. As a control, anon-replication competent adenovirus vector dl312 containing no insertedgene was used.

Human large bowel cancer cell SW620 was transplanted subcutaneously intothe back of 5-6 week-old nude mice at 5×10⁶ cells/mouse. When the tumorbecame approximately 5-6 mm in diameter, 2×10⁷ PFU of dl312/day and4×10³ PFU of TRAD/day were injected intratumorally and locally forconsecutive three days. The axes of each tumor were measured in the samemanner as described above, followed by calculation of the estimatedtumor weight. The results are shown in FIGS. 8 and 9 (the term “Mock”appealing in these Figures represents control to which PBS (phosphatebuffered saline) was administered).

Administration of Ad-p53 at 3×10⁸ PFU and 1×10⁹ PFU inhibited the growthof H358 tumor significantly (p<0.05). However, administration of Ad-p53at 1×10⁸ PFU revealed no significant growth inhibition (FIG. 8).Administration of dl312 (control) indicated no influence upon tumorgrowth.

Intratumoral administration of TRAD at 4×10³ PFU, which is extremelylower than the concentration of Ad-p53 that showed antitumor effect,inhibited the growth of SW620 tumor significantly (p<0.05).Administration of dl312 (control) indicated no influence upon tumorgrowth.

From what have been described above, it is understood that the virus ofthe present invention grows efficiently in cancer cells and brings deathto them. Further, since the virus of the invention has the ability togrow, it is capable of manifesting potent anti-cancer effect even at alow concentration. Thus, it is also possible to reduce side effect byadministering the virus at a low concentration.

1. A polynucleotide comprising a promoter from human telomerase and atleast one E1 gene.
 2. The polynucleotide according to claim 1, whereinthe E1 gene is an adenovirus-derived E1 gene.
 3. The polynucleotideaccording to claim 1, wherein the promoter from human telomerase ishTERT.
 4. The polynucleotide according to claim 1, wherein the E1 genecomprises an E1A gene, an IRES sequence and an E1B gene in this order.5. A virus comprising the polynucleotide according to claim
 1. 6. Thevirus according to claim 5, wherein the virus is an adenovirus.
 7. Ananticancer agent comprising the virus according to claim 5 as an activeingredient and a pharmaceutically acceptable carrier, excipient ordiluent.
 8. A method of treating cancer, comprising using the virusaccording to claim
 5. 9. The method according to claim 8, wherein thecancer is at least one cancer selected from the group consisting ofstomach cancer, large bowel cancer, lung cancer, liver cancer, prostatecancer, pancreas cancer, esophagus cancer, bladder cancer,gallbladder/bile duct cancer, breast cancer, uterine cancer, thyroidcancer and ovarian cancer.
 10. The method according to claim 9, whereinthe cancer is at least one selected from the group consisting ofosteosarcoma and brain tumor.
 11. A method of treating cancer,comprising using the anticancer agent according to claim 7.